Project Abstract: ( 30 lines maximum) The primary cilium is a microtubule-based structure that protrudes from the surface of nearly every cell type in the mammalian body. At the base of the cilium is a complex of proteins forming the transition zone (TZ), a diffusion barrier separating the cilium from the cytoplasm. Correct separation of these domains is important for establishing the cilium as a specialized sensory and signaling compartment. How proteins are transported into and out of the cilium across the TZ is poorly understood but appears to involve a complex of eight proteins that constitute the BBSome. Mutations disrupting these structures, and others related to primary cilia result in a spectrum of human diseases collectively called ciliopathies. These disruptions manifest in a plethora of physiological and developmental disorders that are highly variable. The primary goals of my study are to elucidate the mechanism by which the TZ and BBSome collaboratively work to regulate primary cilium functions important for normal cardiac development in mice, and to elucidate ciliary related regulation of Congenital Heart Defects (CHD). In the United States, CHD is the leading cause of birth-defect associated illness and death in infants. Studies from a genetic screen orchestrated to identify CHD-related genes identified 34 that were associated with the cilium out of 61 total CHD associated genes. Furthermore, studies that have specifically removed the primary cilium from the developing heart have reported CHD related abnormalities. These data strongly support the premise of this application that the primary cilium plays an extensive, but not yet understood, role in CHD pathogenesis. We have previously identified identified genetic interactions between the TZ component, Nphp4, and the BBSome component, Bbs5, through studies in C. elegans. The goal of my study is to extend these studies into a mammalian system and to define the contribution that Nphp4 and Bbs5 have during heart development. My preliminary data indicate that Nphp4; Bbs5 double mutant mice are embryonic lethal between embryonic day 16.5 and birth, while each of the single mutants is viable. My preliminary studies indicate that the lethality is likely associated with critical events disrupting cardiac development. Based on my preliminary data, I hypothesize that genetic interactions between Nphp4 and Bbs5 are necessary for proper cilia function and cardiovascular development, likely through altered regulation of transport of proteins into and out of the cilium. Thus, the data generated from this study will provide critical new understanding of how the primary cilium regulates early cardiac development and how cilia dysfunction contributes to CHD.